Many proteins involved in the vesicle trafficking, neurotransmitter release, and other vesicle membrane fusion events have been identified in the past several years. Interactions among some of these proteins have been defined and molecular models of the exocytotic machinery have been proposed, however, it is still not clear how these proteins and protein- protein interactions are regulated, or the sequences of these interactions that they play in the exocytotic pathway. Munc18a, and its homologues, have been shown to be essential to exocytosis and its specific binding with syntaxin, although it appears to be inhibitory to exocytosis, has been suggested to play a role in the regulated vesicle release pathway. Recently, a novel family of Muncl8a-interacting (Mint) proteins has been identified. The specific interactions and co-expression of these Mint proteins and Muncl8a suggest a regulatory role of these Mint proteins in the exocytotic pathway, however, the regulation of Mint-Munc18a interactions and the functional consequence(s) of these interaction have not been explored. Our preliminary data show promising findings suggesting that Mint proteins may suppress the secretory responsiveness through a Munc18a-related pathway. The focus of this proposal is to consolidate these findings, define the functional domains of Mint proteins involved, understand the mechanism of the action, and explore the role of Munc18a in the regulatory effect of Mint proteins. A multidisciplinary approach, which combines molecular biology, biochemistry, immunology, pharmacology, and electrophysiology, will be used in the proposed investigations. These studies will add new insights in the regulation of exocytotic pathway and lead to a new and more informative model for vesicle trafficking.